The Epithelial-Mesenchymal Transition (EMT) Regulatory Factor SLUG (SNAI2) Is a Downstream Target of SPARC and AKT in Promoting Melanoma Cell Invasion (original) (raw)
Figure 4
SPARC-mediated SLUG expression and promotion of melanoma cell migration is PI3K/AKT dependent.
(A) Influence of PI3K/AKT and BRAFV600E signaling on SLUG expression: left panel, 501mel cells control (cl. CTRL) or overexpressing SPARC (cl. SPARC) were treated with DMSO or 10 mmol/L AKT inhibitor IV (AIIV). Immunoblots show the phosphorylation status of AKT (Ser473) and GSK3β (Ser9) and expression levels of SPARC-Myc transgene, AKT, GSK3β and SLUG. Right panel, 501mel cl. SPARC cells were treated with DMSO or 10 mmol/L AIIV, LY294002 or PLX4720 for 3 hours. Immunoblots show the phosphorylation status of MAP kinases ERK and AKT and expression levels of SLUG. HSP60 was used as a loading control. (B) Influence of PI3K/AKT signaling on cell migration: serum-stimulated cell migration was analyzed using Boyden chamber assays in control and SPARC 501mel cells treated with DMSO or 10 mmol/L AIIV or LY294002 as indicated. Results are expressed in percent of control. Columns, means of triplicates from two independent experiments; error bars, SD. *P<0.05 (Student’s test). Representative images of lower surface of membranes are shown. (C) Expression of Myr-AKT protects from SLUG decrease after SPARC depletion: 501mel cells were infected with a control empty adenovirus (AdCMV) or with adenovirus encoding the constitutively active mutant Myr-HA-AKT1 (AdAKTca) and transfected 6 hours later with 50 nM siCTRL or siSPARC for 3 days. Cells were harvested and proteins were analyzed by immunolotting using the indicated antibodies. (D) Expression of Myr-AKT rescues migratory defects of SPARC-depleted cells: serum-stimulated cell migration was analyzed using Boyden chamber assays in 501mel cells infected with AdCMV or AdAKTca and after depletion for endogenous SPARC as described above. Results are expressed in percent of control. Columns, means of triplicates from two independent experiments; error bars, SD. P<0.05; NS, not significant (Student’s test). Representative images of lower surface of membranes are shown.